The induction of systemic unresponsiveness to orally administered soluble proteins and other antigens, termed oral tolerance, has recently taken on added significance for possible use in clinical applications to prevent autoimmune disease. However, when we consider oral vaccines for effective immunity through the Common Mucosal Immune System, the induction of oral tolerance would not be beneficial since ideal mucosal vaccines should result in both mucosal and systemic immunity. Thus, one can only accomplish oral tolerance to self antigens versus immunity to foreign vaccine proteins when a better understanding of the fundamental mechanisms of oral tolerance are set forth. The most compelling work to date suggests that T lymphocytes are the major regulatory cell type responsible for induction of oral tolerance. However, the precise role of CD4+ versus CD8+ T cells is not yet clear. CD4+ T cells can be divided into two broad subsets based upon patterns of cytokines produced. Th1 cells secrete IL-2, IFN-gamma and TNF-beta and induce cell-mediated immunity as well as help for IgG-subclass responses. In contrast, Th2 cells synthesize IL-4, IL-5, IL-6 and IL-10 and regulate IgG1, IgA and IgE responses. We have hypothesized that oral administration of soluble antigens such as diphtheria toxoid (DT) induces unresponsiveness (including anergy) in the CD4+ T cell subset. We further hypothesize that oral DT induces anergic Th1 cells and DT-specific Th2 cells which further suppress the former subset via production of IL-4 and IL-10. This combined suppression accounts for DT-specific systemic unresponsiveness. In contrast, DT- specific Th2-type cells producing IL-5 and IL-6 support IgA anti-DT responses in mucosal effector sites. To test this hypothesis, the following specific aims are proposed. l) Examination of DT-specific Th1 and Th2 cell responses in Peyers patches (PP) and spleen (SP) of mice orally tolerized with DT by using antigen-induced proliferative responses, as well as cytokine-specific ELISPOT, ELISA and reverse transcription (RT)-PCR assays. 2) Usage of anti-cytokine-treated i.e., anti-IFN-gamma, anti-IL-4 or anti-IL-10, as well as IFN-gamma and IL-4 knockout mice to determine the exact role of in vivo Th1 and Th2 cells in oral tolerance. 3) Involvement of CD8+ T cells for the induction of oral tolerance since recent work has shown that this subset contains TGF-beta producing cells which induce bystander suppression. To directly address this issue, transgenic anti-Lyt-2 or anti-Lyt-3 as well as beta-2 microglobulin (class I) knockout mice will be used in these studies. 4) Role of intraepithelial lymphocytes (IELs), especially the gamma/delta TCR+ subset, for maintenance of mucosal IgA responses in a state of oral tolerance. 5) Test whether the mucosal adjuvant cholera toxin (CT) can reverse an existing state of oral tolerance by enhancing DT-specific Th2 cells. The first 3 specific aims are focused on cellular and molecular mechanisms involved in systemic unresponsiveness, while the last 2 focus more on how the mucosal IgA response is maintained in the presence of systemic unresponsiveness. Further, we will examine the mucosal adjuvant CT to determine if systemic unresponsiveness can be reversed. To accomplish these goals, we will use the most modern molecular approaches including specific knockout and transgenic animals which allow in vivo studies of T cell mediated oral tolerance.